Carotenoids are pigments that are ubiquitous throughout nature and synthesized by all oxygen evolving photosynthetic organisms, and in some heterotrophic growing bacteria and fungi. Carotenoids provide color for flowers, vegetables, insects, fish and birds. Colors of carotenoid range from yellow to red with variations of brown and purple. As precursors of vitamin A, carotenoids are fundamental components in human diet, playing an important role in human health. Industrial uses of carotenoids include pharmaceuticals, food supplements, animal feed additives and colorants in cosmetics to mention a few.
Because animals are unable to synthesize carotenoid de novo, they must obtain them by dietary means. Thus, manipulation of carotenoid production and composition in plants or bacteria can provide new or improved source for carotenoids.
Carotenoids come in many different forms and chemical structures. Most naturally occurring carotenoids are hydrophobic tetraterpenoids containing a C40 methyl-branched hydrocarbon backbone derived from successive condensation of eight C5 isoprene units (IPP). In addition, novel carotenoids with longer or shorter backbones occur in some species of nonphotosynthetic bacteria. The term “carotenoid” includes both carotenes and xanthophylls. A “carotene” refers to a hydrocarbon carotenoid. Carotene derivatives that contain one or more oxygen atoms, in the form of hydroxy-, methoxy-, oxo-, epoxy-, carboxy-, or aldehydic functional groups, or within glycosides, glycoside esters, or sulfates, are collectively known as “xanthophylls”. Carotenoids are furthermore described as being acyclic, monocyclic, or bicyclic depending on whether the ends of the hydrocarbon backbones have been cyclized to yield aliphatic or cyclic ring structures (Armstrong, G., Comprehensive Natural Products Chemistry, Elsevier Press, volume 2, pp. 321–352. (1999)).
Carotenoid biosynthesis starts with the isoprenoid pathway to generate the C5 isoprene unit, isopentenyl pyrophosphate (IPP). IPP was condensed with its isomer dimethylallyl pyrophophate (DMAPP) to C10 geranyl pyrophosphate (GPP) and elongated to C15 farnesyl pyrophosphate (FPP). FPP synthesis is common in both carotenogenic and non-carotenogenic bacteria. Subsequent enzymes in the carotenoid pathway generate carotenoid pigments from the FPP precursor and can be divided into two categories: carotene backbone synthesis enzymes and subsequent modification enzymes. The backbone synthesis enzymes include geranyl geranyl pyrophosphate synthase (CrtE), phytoene synthase (CrtB), phytoene dehydrogenase (CrtI) and lycopene cyclase (CrtY/L), etc. The modification enzymes include ketolases, hydroxylases, dehydratases, glycosylases, etc.
Lycopene cyclases are a class of enzymes responsible for catalyzing the formation of cyclical carotenoids from lycopene (ψ,ψ-carotene), an acyclic symmetrical carotenoid. Lycopene cyclases catalyze the formation of ionone rings from the ψ end groups found on lycopene (FIG. 1).
Two types of lycopene cyclases (β-cyclases and ε-cyclases) have been reported (Cunningham et al., Plant Cell. 8:1613–1626 (1996)). All previously described lycopene β-cyclases catalyze the formation of β-ionone rings from ψ end groups found on acyclic carotenoids such as lycopene (ψ,ψ-carotene), usually resulting in a symmetrical bicyclic product such as β-carotene. The lycopene ε-cyclases, usually found in plants, catalyze the formation of ε-ionone rings from the ψ end groups. Most lycopene ε-cyclases catalyze formation of the asymmetric monocyclic δ-carotene (ψ,ε-carotene). A lycopene ε-cyclase from lettuce catalyzes the formation of bicyclic ε-carotene (ε,ε-carotene) (Cunningham et al., PNAS, 98:2905–2910, (2000)). The difference between the β-ionone and ε-ionone ring structure is based on the location of the double bond within the 6-member ring.
The known lycopene β-cyclases function symmetrically on lycopene, creating symmetric bicyclic β-carotene through a monocyclic γ-carotene intermediate. A lycopene β-cyclase isolated from Pantoea ananatis was reported to produce bicyclic β-carotene via a 2-step reaction involving γ-carotene as the intermediate (Schnurr et al., Biochem J. 315: 869–874 (1996)).
Monocyclic carotenoids are sometimes present as part of the mixture during bicyclic carotenoids synthesis. Certain methods such as using a β-cyclase mutant with decreased activity or a partial inhibition of the β-cyclase could be used to enrich for the monocyclic carotenoids in the mixture. Isolation and purification of monocyclic carotenoids from the mixture of carotenoids derived from β-cyclases often requires a significant investment in time and resources. No prokaryotic lycopene β-cyclase has been proven to selectively produce only monocyclic carotenoids.
A monocyclic β-cyclase pathway has been proposed to exist in the yeast Phaffia rhodozyma by enzyme inhibition experiments (An et al., J. Biosci. Bioeng. 88(2): 189–193 (1999)). However, the monocyclic carotenoids produced in Phaffia were a minor component (<20%) of the total carotenoid mixture, and the presence of an enzyme that was selective for the production of monocyclic carotenoids was not taught.
Plant lycopene ε-cyclases have been shown to primarily make monocyclic carotenoids, and only make ε-ionone ring structures (Cunningham et al., Plant Cell. 8:1613–1626 (1996) and Cunningham F. and Sun Z. U.S. Pat. No. 5,744,341).
The genetics of carotenoid pigment biosynthesis are well known (Armstrong et al., J. Bact. 176: 4795–4802 (1994); Annu. Rev. Microbiol. 51:629–659 (1997)). This pathway is extremely well studied in the Gram-negative, pigmented bacteria of the genera Pantoea, formerly known as Erwinia. In both E. herbicola EHO-10 (ATCC 39368) and E. uredovora 20D3 (ATCC 19321), the crt genes are clustered in two operons, crt Z and crtEXYIB (U.S. Pat. No. 5,656,472; U.S. Pat. No. 5,545,816; U.S. Pat. No. 5,530,189; U.S. Pat. No. 5,530,188; U.S. Pat. No. 5,429,939). Despite the similarity in operon structure, the DNA sequences of E. uredovora and E. herbicola crt genes show no homology by DNA-DNA hybridization (U.S. Pat. No. 5,429,939).
Although the genes involved in carotenoid biosynthesis pathway are known in some organisms, genes involved in carotenoid biosynthesis in Rhodococcus and Deinococcus bacteria are not described in the existing literature. Analytical characterization of carotenoid pigments from several strains of Rhodococcus was conducted (Ichiyama et al., Microbiol. Immunol. 33:503–508 (1989)). However, the analytical characterization did not attempt to characterize the enzymes responsible for the “γ-carotenoid-like” compounds reported.
The problem to be solved therefore is to provide methods and materials useful for the selective production of asymmetric carotenoids containing a single β-ionone ring. Applicants have solved the stated problem by isolating and characterizing genes encoding for a novel lycopene β-cyclase (crtL), isolated from both Rhodococcus and Deinococcus, which encode polypeptides that selectively produces monocyclic (β-ionone ring) carotenoids without significant bicyclic carotenoid synthesis.